The present investigation was carried out to study the bioactivity of four Olea europaea cultivars (Chemlali; CHM, Manzanilla; MAZ, Picaul; PIC and Tofahi; TOF) leaves aqueous extract (OELAE) as well as crude powder (OELCP) on germination efficiency, physiological, biochemical and molecular parameters of Hordeum vulgare. The allelopathic effect varied according to treatment and cultivar. An increase in total seedling dry weight (TSDW) was achieved with all tested concentrations (0.25, 0.5, 0.75, 1, 1.25 and 1.5%) of the four cultivars except TOF cultivar. Leaves of the donor species attained values of oleuropein varied from 35 to 67 mg g^-1 dry weight depending upon the cultivar which was highly correlated with the level of antioxidants activity of germinated H. vulgare. Chlorophyll a (Chl a) recorded a marked increase with the increase in OELCP except for TOF. Noticeably, chlorophyll b (Chl b) and carotenoids (Carot) has shown a disturbance in their concentrations with both treatments and cultivars. The CHM (low oleuropein content) cultivar elicits high percentage of genomic template stability (GTS %) in H. vulgare for the two-affecting concentration measuring about 86% and 82% respectively. To go through with this, TOF cultivar (high oleuropein content) achieved the lowest percentage of genomic template stability (GTS %) in the two-affecting concentrations of about 36% and 32%. Finally, we can conclude that if the growing barley is planting for herbal medicine purposes as antioxidant it prefers to be cultivated under CHM and MAZ olive trees and avoided cultivation under the two other cultivars; PIC and TOF.